Euphytica 35 (1986)
ISOLATION OF A PURE THORNLESS LOGANBERRY BY MERISTEM TIP CULTURE
H.K. HALL1, M. H. QUAZI2 and R. M. SKIRVIN3
Crop Research Division; DSIR, Private Bag, Christchurch, New Zealand
Received 24 February 1986
|1 Present address:
DSIR, Riwaka Research Station, RD 3, Motueka, New Zealand.
2 Present address: Departement of Plant Breeding and Genetics, N.W.F.P. Agricultural University, Peshawar, Pakistan.
3 Present address: University of Illinois, Department of Horticulture. 7707 So. Orchard, Urbana, Illinois 61801, USA.
Rubus, blackberry, raspberry, hybridberry, Loganberry, chimera, in vitro, plant breeding, tissue culture, thornless
LIST OF TERMS: BAP = 6-benzylaminopurine; GA) = gibberellic acid; NAA = naphthalene acetic acid: hybridberry = polyploid bramble interspecific hybrids; MS = MURASHIGE & SKOOG (1962) high mineral salt medium; TL = 'Thornless Loganberry'; TLtc = tissue culture-derived (non-chimeral) 'Thornless Loganberry'.
'Thornless Loganberry' (TL) is a periclinal chimeral blackberry in which a layer of mutant (thornless) epidermis surrounds a core of wild-type (thorny) tissue. Due to its chimeral arrangement, TL produces thorny adventitious root cuttings and thorny offspring. To separate the chimera into its components parts, meristems of TL were grow in vitro on modified Murashige and Skoog medium to yield callus and adventitious shoots. One of these shoots has survived, flowered, and produced thornless offspring from seed. The importance of this non‑chimeral TL is discussed.
'Thornless Loganberry', (TL) (Rubus sp.) (2n = 6x = 42) is a fully fruitful sport of the 'Logan' blackberry. It was discovered in 1929 and distributed as 'Bauer Thornless' (JENNINGS, 1981). Both thorny and thornless types have been propagated asexually and by seeds to result in a 'considerable admixture of inferior [thorny] seedlings and virus‑infected plants' (JENNINGS, 1981). From these mixtures both thorned and thornless Loganberry clones have been reselected ('LY59' and 'L654'. respectively) (BEAKBANE, 1935; BEAKBANE & LABERN, 1960; WAY, 1966).
Unfortunately, due to chimerism, TL thornlessness genes are not available to the geneticist (DARROW, 1937). TL is a periclinal ('hand-in-glove') chimera in which a layer of mutant (thornless) epidermis (L1) completely encloses a core of wild-type (thorny) tissue (L2+L3). Since thorns (technically known as prickles) have an epidermal origin, the entire plant appears to be thornless.
Under some situations the thornless condition is stable, but when the plant develops adventitious shoots, from roots (L3) or when it freezes to the ground, the plant regenerates itself from internal tissues to yield thorny shoots (DERMAN, 1947; DARROW, 1955). In addition, Rubus gametes also develop from internal (L2) regions of the shoot and therefore produce thorny seedlings. DARROW (1937) reported only thorny offspring from selfed TL flowers. Similar observations have been made by the senior author in selfed and hybrid progenies involving TL. HULL & BRITTON (1958) reported a single thornless seedling from a TL x Eldorado (8 x ) cross.
MCPHEETERS & SKIRVIN (1983) have reported a tissue culture system where 'Thornless Evergreen' (a periclinal chimera similar to TL) blackberry was separated into its component parts. Among the regenerated plants a large percentage (46.3%) were of epidermal origin. These plants were believed to be pure thornless because they produced thornless rather than thorny adventitious shoots from isolated root segments.
To use the TL thornlessness (gene(s) for bramble improvement, chimeral TL must be separated into its component parts to yield a non-chimeral thornless form.
MATERIALS AND METHODS
Shoot tips of 'TL' ('L654') were gathered from a glasshouse grown plant at Lincoln. New Zealand, in November, 1981. These tips were disinfested with 70 per cent ethanol for 30 seconds: 0.32 per cent sodium hypochlorite for 20 minutes and three washes in sterile water. The meristem (0.5 mm) was excised and transferred to ANDERSON'S (1980) medium. The meristems developed calli which were sub-cultured regularly. In May, 1982, the calli were moved to a new basic medium which consisted off 1/2 strength MS (1962) macro salts, full strength MS micro salts and iron: the vitamins of Gamborg's B5 medium (Gamborg, et al.. 1968): sucrose (30 g/litre): and agar (0.6%). This medium was supplemented with kinetin (0.5 mg/litre) and NAA (0.1 mg/litre) and/or various levels of GA (1, 5, 10, or 20mg/litre) and BAP (0.05, 0.1, 0.5 or 2.05 mg/litre). The pH was adjusted to 5.5 prior to autoclaving. The media were dispensed into 100 ml erlenmeyer flasks (25 ml/flask) and sterilized by autoclaving at 121 °C for 15 min.
The cultures were maintained at 22 ± 3 °C under diffuse light, 16 hr day length, for the first four weeks, when light intensity was increased to 10,000 lux. Callus growth and differentiation were monitored on a regular basis. Callus was subcultured onto identical media. Rooted adventitious shoots were moved directly to soil and placed under intermittent mist within a plastic‑enclosed humidity chamber in a glasshouse. As the plants hardened, they were removed from the mist and grown to maturity.
To investigate the internal genotype of regenerates, root cuttings were obtained, placed in sand, and stratified for 2 months @ 4 ± 1 °C. They were then brought into a glasshouse for adventitious shoot evaluation.
The mature pot-grown plants were maintained in a screenhouse where they received natural chilling. In the spring of 1984, the plants were forced to flower in the glasshouse and selfed to investigate the possibility of the thornless character being expressed sexually. Seeds were harvested, extracted, and dried. They were scarified and stratified according to the procedures followed by the Scottish Crop Research Institute (personal communication, D. H. Jennings): seeds were scarified in concentrated sulphuric acid for 30 minutes, rinsed in running water, and immersed in 1% calcium hypochlorite (w/v) which was dissolved in a saturated solution of calcium hydroxide for one week. At the end of that period, the seeds were washed in running water and immediately mixed with sand and sown into a 15 cm diameter pot filled with 10 cms of ground pumice and 2.5 cms of peat‑sand. The pots were watered and placed in sealed plastic bags. The pots were moved to a screenhouse and covered by shade cloth to prevent heat accumulation. The seeds were stratified by natural winter chilling. In the early spring, the pots were transferred to a heated glasshouse for germination. Germinated seeds were pricked out and transferred to individual pots. When large enough, they were evaluated for the thorned or thornless condition.
|Fig. 1. Callus which has developed from a meristem of 'Thornless Loganberry'.||Fig. 2. A rooted adventitious shoot of 'Thornless Loganberry' which has developed from meristem-derived callus.|
|Fig. 3. A pure thornless (non-chimeral) 'Loganberry' that has flowered and been used for hybridisation.||Fig. 4. A thornless (right) and thorny (left) seedling derived by self-pollinating a pure thornless (non-chimeral) 'Loganberry'.|
Most media supported TL callus proliferation, but only the basic medium supplemented with GA3 (5.0 mg/litre) and BAP (0.05 mg/litre), developed rooted shoots (Figure 1 and 2). A total of 3 shoots developed. Two of these died when transferred to soil. The remaining plant, which was totally thornless, survived grew well, and eventually flowered (Fig. 3). This tissue culture-derived plant (TLtc was fully male and female fertile and indistinguishable from TL (Fig. 3). No adventitious shoots were obtained from the stratified root cuttings.
Three hundred selfed seed were treated for germination; 100 of these germinated and 100 grew enough to be evaluated. Among the seedlings both thorny and completely thornless offspring (63%) were found (Fig. 4).
'Loganberry' has been used extensively as a parent for hybridberry improvement because of its superior fruit qualities and general combining ability (personal communication, F. J. Lawrence, USDA. Corvallis, Oregon, USA; DARROW, 1973). Unfortunately, TL normally produces only spiny offspring. The one case of a thornless seedling from TL arose from a cross of TL x Eldorado (8 x) (HULL, 1958). Since this plant produced thornless root suckers, Hull suggested that the seedling was an apomict. However, it is unlikely that this was the case because the thornless character of TL normally is epidermal, not internal, and sexual organs, including the nucellus, are of internal origin (DERMAN, 1947). The thornless apomict could only have arisen from a region of sectoral, not periclinal, chimeral arrangement. However, sectoral chimeras could arise unobserved in TL, since both chimeral and pure epidermal tissues can be fully fruitful and morphologically indistinguishable. In contrast, the epidermal-derived shoots of the similar chimeral cultivar, 'Thornless Evergreen', are relatively sterile (MCPHEETERS 1985) and morphologically distinct (MCPHEETERS & SKIRVIN, 1983). No references to Hull's plant being used for breeding have been published and it is presumed to have died.
When TLtc was selfed it produced both thornless and thorny seedlings (Fig. 4). The relatively low germination percentage (36.7%) was not atypical for Rubus (KE et al.. 1985). The percentage of thornless offspring was high enough (63%) to suggest dominant inheritance, however, the genetics of 'Loganberry' thornlessness require further investigation.
Thornlessness is of particular value for hand harvesting brambles; pickers harvest fruit faster and more thoroughly when they expect no damage from thorns. Thornlessness is also important for mechanical harvesting. While thorns do not damage machinery, the vigorous activity of mechanical harvesting can dislodge thorny plant parts which contaminate harvested fruits and/or processed products (personal communications, various Oregon caneberry growers and processors). In addition, thorn‑punctured fruits result from mechanical harvesting. Punctured drupelets, which are found on both harvested and unharvested fruits, bleach, leak juice, and decay rapidly. In the future, processors are expected to make increased demands for thornless polyploid hybridberry cultivars. 'Loganberry' thornlessness genes may help geneticists supply these thornless cultivars.
In conclusion, the isolation of a pure thornless 'Loganberry' (TLtc) demonstrates the usefulness of tissue culture for the improvement of standard fruit cultivars. Because TLtc is fruitful, it may have direct use as a new thornless 'Loganberry' cultivar in regions with severe winters. Since TLtc passes its thornless character through the sexual cycle, the thornlessness of TL can now be used to produce thornless hybridberries.
This research was paid for in part by funds provided by the New Zealand Department of Scientific and Industrial Research (DSIR) and the University of Illinois Experiment Station (Urbana, IL, USA). Special tanks to Fred Braam, Jeanette Dodson and Michelle Williams for technical assistance.
"McPheeters and Skirvin (70) proliferated over 900 plants of Rubus laciniatus 'Thornless Evergreen' from shoot tips, obtaining 53% thornless chimeral plants and 47% dwarf, pure thornless plants. The mutant layer in 'Thornless Evergreen' resides in the L.I such that the derivatives are unable to produce prickles as are the derivatives of L.II and L.III. McPheeters and Skirvin were surprised not to have obtained a certain proportion of nonchimeral thorny shoots (from endogenous L.II and/or L.III derivatives) and concluded that the tissue culture conditions must not have been conducive for such bud formation. From the fact that thorny shoots are more vigorous-growing than thornless (in the field), it is surprising that at least some did not arise. Under their conditions, L.I participated in all shoot formation, axillary and adventitious, while L.II and L.III evidently were only involved during axillary bud formation. The possibility exists that epidermal tissue in direct contact with the medium may have responded so rapidly in forming shoots that the endogenous tissues were left behind."
Pakistan Journal of Biological Sciences 6(19): 1644-1648, 2003
Separation of Thornless Rose Chimeras into Their (Rosa sp.) Consistent Genotypes in vitro
Fatih Au Canli and Robert M. Skirvin Department of Horticulture, Faculty of Agriculture, Suleyman Demirel University, Central Campus 32260 ISPARTA, Turkey 'University of Illinois at Urbana-Champaign, Urbana IL USA
Abstract: To isolate a pure thornless rose, a thornless sport of Rosa multiflora ('Fairmount 1') was established in tissue culture. To determine whether the sports were chimeral and consisted of both thorny and thornless tissue, the regenerants obtained in the tissue culture studies were assessed for the thorny or thornless condition both in vitro and ex vitro. Chimeral plants were expected to yield both thorny and thornless regenerants. Among these regenerants we expected to obtain a pure thornless rose. All tissue culture experiments with 'FM1' yielded both thorny and thornless regenerants, clearly demonstrating that 'FM1' is chimeral. TDZ significantly affected segregation both in vitro and ex vitro. The percentage of thorny plants was highest at 14.4 µM TDZ and lowest with control (0 µM TDZ). The linear relationship between increasing TDZ concentrations and percentage of thorny plants at vitro was significant. These results show rose chimeras can be separated into their component genotypes by growing them on media with high concentrations of TDZ (3.6, 5.4, 7.2 and 14.5 µM TDZ). Also among 240 plants from the TDZ experiments, 21 plants were classified as thornless after a seven months in the greenhouse.
CybeRose note: TDZ = thidiazuron